Reconstruction of Mammalian Cells from Nuclear and Cytoplasmic Components Separated by Treatment with Cytochalasin B

Enucleation techniques combining mild centrifugation in the presence of cytochalasin B permit cells to be separated into nuclear fragments (karyoplasts) and cytoplasmic fragments (cytoplasts). These fragments, though stable for a short time, will ultimately degenerate by the procedures described in this report. One can, however, fuse cytoplasts to karyoplasts by using polyethylene glycol and obtain viable reconstituted cells whose properties may be useful for understanding some aspects of the nuclear-cytoplasmic interactions associated with tumorigenicity and steroidogenesis. However, the presence of cybrids, hybrids, and parental whole cell contaminants along with the reconstituted cell population make it necessary to have genetic markers that reside in both the nucleus and cytoplasm in order to preferentially identify reconstituted cells derived from a karyoplast fused to a cytoplast. By utilizing the Y-1 cell line, which is tumorigenic and responds to corticotropin b secreting steroids, and the AMT-BU-A1 (AMT) cell line, which is nontumorigenic and does not respond to corticotropin but has a nuclear marker, BrdUrdr, anda cytoplasmic marker, CAPrF we have reconstituted cells containing Y-1 karyoplasts and AMT cytoplasts. In this report we extend our previous techniques by describing an identification procedure that allowed us to isolate cells reconstituted from AMT karyoplasts fused to Y-1 cytoplasts. The results of these experiments support the concept that with these cell lines the nucleus (karyoplast) is ultimately sufficient to control the phenotypic expression or suppression of tumorigenicity and steroidogenesis.Recent technological advances have allowed investigators to remove the nucleus (karyoplast) from one cell and place that karyoplast into another enucleated cell (cytoplast) (1). The properties of such reconstituted cells may be a potentially valuable tool useful in extending our understanding of nuclear-cytoplasmic interactions. This reconstitution procedure begins by centrifuging monolayers of cells in the presence of cytochalasin B, which results in the separation of the cells into two fragments. One of the fragments contains the nucleus surrounded by variable amounts of cytoplasm and an intact plasma envelope and has been termed karyoplast (2, 3); the other fragment is composed of t90-95% of the cell's original cytoplasm and has been termed cytoplast (2, 3). These fragments may be fused together by using Sendai virus or polyethylene glycol; this process is called cell reconstitution. Because neither of these procedures (enucleation or reconstitution) is 100% efficient, it is necessary to have some way to identify or select reconstituted cells from contaminating parental whole cells, cybrids (the result of the fusion of a cytoplast to a whole cell) (4), and hybrids (one whole cell fused to another whole cell). Previous techniques have made use of physical markers such as latex spheres or genetic markers, such as resistance to bromodeoxyuridine (BrdUrd) and chloramphenicol (CAP), to ide...

mentioning

confidence: 99%

mentioning

confidence: 99%

Alternative method for identifying reconstituted cells

Shay¹,

Clark

1980

“…Some of the mouse polypeptides detected at the earliest time points studied were not among the major polypeptides synthesized by the parental A9 cells. By about 48 hr after fusion, the pattern of polypeptides produced by reconstituted cells was almost indistinguishable from that of the nuclear donor parent cells.Cytoplasts and karyoplasts prepared from animal cells by cytochalasin-induced enucleation (1, 2) can be fused together to reconstitute whole viable cells (3)(4)(5). If the fragments are from two different cell types, cells containing single nuclei within foreign cytoplasm can be constructed (6, 7).…”

mentioning

confidence: 99%

“…Cytoplasts and karyoplasts prepared from animal cells by cytochalasin-induced enucleation (1, 2) can be fused together to reconstitute whole viable cells (3)(4)(5). If the fragments are from two different cell types, cells containing single nuclei within foreign cytoplasm can be constructed (6, 7).…”

mentioning

confidence: 99%

Identification of key regulated events early in the life of hybrid animal cells constructed by nuclear transplantation.

Hightower¹,

Bruno²,

Lucas³

1983

Reconstituted cells were constructed by fusion of cytoplasts from the human diploid fibroblast cell strain Detroit 532 and karyoplasts from the mouse fibroblast cell line A9. Several cellular properties were examined during the first 48 hr after nuclear transplantation. (i) The overall morphology of the cells originally resembled that of the cytoplasmic donor, Detroit 532, but rapidly changed to approximate that of the nuclear donor, A9. However, definitive changes in the microfilament structure of the reconstituted cells were not seen until 24-48 hr after fusion. These observations support the idea that the presence or absence of an ordered array of microfilament bundles is not the sole determinant of cell shape. (ii) Although cytoplasts and karyoplasts were prepared from cultures of randomly growing cells, the first division of reconstituted cells occurred in a synchronous manner. However, the initiation of DNA synthesis was not synchronized. It thus appeared that, in their first cell cycle, the cells had a G2 period of variable length. The results further suggest that the cytoplasm of interphase fibroblasts contains the material necessary to initiate or support DNA synthesis in a transplanted nucleus but not entry into mitosis. (iii) A two-dimensional gel electrophoretic analysis of polypeptide synthesis in reconstituted cell cultures showed that synthesis directed by transplanted mouse nuclei could be detected as early as 3-6 hr after fusion. Some of the mouse polypeptides detected at the earliest time points studied were not among the major polypeptides synthesized by the parental A9 cells. By about 48 hr after fusion, the pattern of polypeptides produced by reconstituted cells was almost indistinguishable from that of the nuclear donor parent cells.Cytoplasts and karyoplasts prepared from animal cells by cytochalasin-induced enucleation (1, 2) can be fused together to reconstitute whole viable cells (3)(4)(5). If the fragments are from two different cell types, cells containing single nuclei within foreign cytoplasm can be constructed (6, 7). By judicious choice of the parental cell types and the use of a variety of staining techniques, such reconstituted cells can be produced in large numbers and definitively identified immediately after fusion (8-11). These cells, having a minimal contribution of cytoplasm from the nuclear donor cell type (12-15), would appear to be excellent systems for investigating the interaction of cytoplasm and nucleus in determining a variety of cellular properties and events, including cell shape and cytoarchitecture and genome replication and expression. However, analyses of both reconstructed and cybrid cells performed so far have indicated that, except for a few instances (16-21), exposure to a foreign cytoplasm has little long-term effect on a transplanted nucleus. In this report it is shown that, in most respects, cells constructed by the method of nuclear transplantation are indistinguishable from the nuclear donor parental cell type as early as 48 hr after fusion....

“…The 3T3 cells that served as nuclear donors were grown in medium containing approximately 106 large latex spheres per ml (1.0,gm diameter) (Duke Standards, Palo Alto, CA). Each cell took in by phagocytosis approximately 10 spheres, as previously described (4). During the enucleation, almost all of the spheres remained with the cytoplasts and were used as morphological markers for whole 3T3 cells that detached during enucleation.…”

mentioning

confidence: 99%

Selection of reconstituted cells from karyoplasts fused to chloramphenicol-resistant cytoplasts

Shay¹

1977

Murine Balb/3T3 and murine A-MT-BU-A1 mammary tumor cells were separated in the presence of cytochalasin B into enucleated cytoplasmic components (cytoplasts) and nucleated subcellular components (karyoplasts). Karyoplasts were derived from 3T3 cells, while cytoplasts were derived from A-MT-BU-A1 cells that were both chloramphenicol-resistant (CAPr) and sensitive to hypoxanthine/aminopterin/thymidine (HATS). CAPr has been shown to be cytoplasmically transmitted (possibly a mitochondrial gene mutation), while sensitivity to medium containing HAT has been shown to be transmitted by the nucleus (i.e., nuclear gene mutation). Such CAPr cytoplasts derived from A-MT-BU-A1 cells were then fused, using polyethylene glycol, to HAT-resistant 3T3 karyoplasts. The mononucleated reconstituted cells produced by such procedures were cloned in medium containing both HAT and CAP. Some of the reconstituted cells survived, because they were resistant to both drugs, while the nuclear and cytoplasmic whole cell contaminants were killed by one or the other of the two drugs. The results of these experiments indicate that reconstituted cells that are derived from two different cell lines are viable, as indicated by their ability for long-term proliferation in culture. Most of the clones derived resembled morphologically the 3T3 nuclear donor parent cells, but some of the clones did not resemble either parental cell line. It is anticipated that such selection techniques will permit more complete analysis of interrelationships between nucleus and cytoplasm.Cytochalasin B can be used to separate animal cells growing in monolayer culture into nuclear [karyoplasts (1, 2) and minicells (3)] and cytoplasmic [cytoplasts (1, 2)] components. As previously reported (2), these cell fragments die within 48-72 hr after separation, but in the past 2 years, workers in several laboratories have reported reconstitution and subsequent viability of mammalian cells by fusing karyoplasts from a specific cell line to cytoplasts from the same cell line (4-6) (homospecific) or to other cell lines (7, 8) (heterospecific). These studies indicate that such reconstitution experiments are feasible, but convincing evidence that such reconstituted cells after several mitotic divisions are not whole cell contaminants that detached during enucleation or that failed to enucleate during the centrifugation is still lacking. The main reason such reconstituted cells have not been satisfactorily identified, as opposed to whole cell contaminants, is due primarily to the lack of both nuclear and cytoplasmic markers that would permit the identification of reconstituted cells, at a time when the properties of such cells may have been modified by the influence of a new combination of nucleus and cytoplasm.